DESCRIPTION (APPLICANT'S ABSTRACT): The long-term goal of our work is to understand the molecular mechanisms that control mRNA-specific rates of translation and degradation. The regulation of mRNA metabolism is a critical step in the control of gene expression. This regulation is often mediated by interactions with elements in the 3' untranslated regions (UTRs) of mRNAs. Several types of RNA binding proteins have been identified that bind these 3' UTR control elements in a sequence-specific manner. However, relatively little is known of the mechanisms by which these RNA binding proteins recognize their 3' UTR elements, or how individual mRNAs are differentially regulated by such interactions. The Puf family of RNA binding proteins is a widely conserved family, of which two members have been shown to bind 3' UTRs to control critical developmental decisions in Drosophila and C. elegans. It has been hypothesized that other members of the Puf family also play a role in the regulation of specific mRNA decay and/or translation rates. By utilizing the genetic and molecular advantages of the yeast Saccharomyces cerevisiae, we identified several mRNAs whose poly(A) tail metabolism and decay are specifically regulated by one or more of the five yeast Puf protein members. Closer examination of the COX1 7 mRNA revealed that a single Puf protein, Puf3p, enhances the rates of deadenylation and decay of this transcript in vivo, and binds to the COX1 7 3' UTR in vitro. Using the COX1 7/Puf3p interaction as a model case of Puf protein regulation, this proposal will focus on understanding the mechanism by which the Puf3p recognizes the COX1 7 3' UTR and promotes rapid deadenylation and decay. We will use a combination of genetic and biochemical approaches to define the RNA-Puf protein interaction and identify additional gene products that are involved in the Puf regulatory pathway. The results of these studies will provide insight into how Puf proteins recognize and regulate mRNA in yeast and other eukaryotes, and into the general principles of 3' UTR control of gene expression.